Studying On The Protocols For Measuring The Interaction Between Ractopamine And Calf Thymus DNA

The investigation of the interaction between small-molecular drug and DNA provides a theoretical basis for people to understand life phenomena and life activities.At present,with the achievement of pharmaceutical science and analytical chemistry,the research of interaction between small-molecular drug and DNA has been developed more in-depth.The interaction between small-molecular drugs and DNA can not only provide new perspectives in life science,but also offer opportunities for chemists and pharmacologists to develop new drugs that can effectively regulate life activities.People have already gotten a deep understanding of the structure of DNA.The interaction between small-molecular drug and DNA will promote people’s understanding of the chemical nature and biological function of DNA.It is of great significance to clarify the small-molecular drug in the mutagenic damage of DNA and protect human health.This study includes the following two parts:（1）With the model of ractopamine and calf thymus DNA（CT-DNA）,a protocol using microdialysis（MD）sampling integrated with flow injection（FI）-chemiluminescent（CL）detection was developed for studying the interaction between small-molecular drug and DNA.After incubating ractopamine with CT-DNA,unbound ractopamine was on-line sampled using a MD probe,followed by being introduced into a FI-CL system for quantitation.The detected concentrations of unbound ractopamine were calibrated with the recovery of the MD probe,and then treated with Klotz analysis and Scatchard analysis to acquire the binding parameters.The MD probe exhibited a mean recovery of 27.3%for ractopamine sampling under the optimal conditions.The binding constants obtained by Klotz analysis and Scatchard analysis were 3.8×10⁶ M^-1and 3.9×10⁶ M^-1.Ractopamine was estimated to have only one type of binding site on CT-DNA.The obtained results demonstrated that the protocol using on-line MD sampling integrated with FI-CL detection is a simple and reliable technique platform for studying the interaction between small-molecular drug and DNA.（2）The interaction mechanism between ractopamine and CT-DNA was studied by UV-Vis spectroscopy and fluorescence spectroscopy.The results were verified with molecular-docking modeling.By UV-vis spectroscopy analysis,we concluded that the groove binding was the main mode of action between ractopamine and CT-DNA.Fluorescence spectroscopy showed that the fluorescence quenching degree of ractopamine was linearly related to the concentration of CT-DNA in a certain range.The fluorescence quenching of ractopamine by CT-DNA was resulted from the static quenching caused by the complex.Molecular docking simulation showed that ractopamine and DNA T-8,A-18,T-19 and T-20 formed a hydrogen bond,and its spatial structure was coincide with the shape of DNA groove.Therefore,it was further demonstrated that the interaction between ractopamine and CT-DNA was groove binding resulted from hydrogen bonding which led to a strong affinity of ractopamine and CT-DNA.This study provides a lot of basic information for the influence of ractopamine on the structure and function of CT-DNA,as well as toxicity mechanism and elimination pathway of adverse reactions.